Inhibitors of Tyrosine Kinase-Dependent Signaling as Anti-Cancer Agents

酪氨酸激酶依赖性信号传导抑制剂作为抗癌药物

• 批准号: 10702292
• 负责人: TERRENCE BURKE
• 金额: $ 45.43万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702292
• 关键词: Advanced Development; Affinity; Aldehydes; Amines; Amino Acid Sequence; Antineoplastic Agents; Area; Benzoic Acids; Binding; Biochemical; C-terminal; Catalytic Domain; Cell division; Clinical Trials; Collaborations; Complex; Crystallization; DNA; DNA Repair Enzymes; Development; Docking; Drug resistance; Elements; Exhibits; Goals; Hot Spot; Hydrolysis; Imidazole; Label; Laboratories; Legal patent; Libraries; Ligand Binding; Ligand Binding Domain; Ligands; Link; Malignant Neoplasms; Mediating; Molecular; Molecular Target; N-terminal; Nitrogen; Oximes; PLK1 gene; Parents; Peptides; Pharmaceutical Preparations; Pharmacology; Phosphopeptides; Phosphoserine; Phosphothreonine; Phosphotransferases; Phthalic Acids; Physiological; Play; Polo-Box Domain; Process; Prognosis; Proteins; Reaction; Reagent; Reporting; Roentgen Rays; Role; Serine; Signal Transduction; Skeleton; Structure; Substrate Interaction; TOP1 gene; Technology; Threonine; Time; Type I DNA Topoisomerases; Tyrosine; Tyrosine Kinase Inhibitor; Up-Regulation; Variant; Work; analog; antagonist; anti-cancer; anti-cancer therapeutic; base; cancer therapy; chemical stability; cytotoxicity; design; improved; inhibitor; inorganic phosphate; nanomolar; overexpression; peptide structure; phosphodiester; polo-like kinase kinase 1; protein kinase inhibitor; protein protein interaction; repaired; small molecule; therapeutic development; therapy development; tool; tyrosyl-DNA phosphodiesterase

defined as a molecular target for anti-cancer therapy development. The Plk1 plays a central role in cell division and upregulation of Plk1 activity appears to be closely associated with aggressiveness and poor prognosis of several cancers. This protein is overexpressed in many cancers and its inhibition can result in antiproliferative effects. Plk1 requires the coordinated actions of both an N-terminal kinase domain (KD), which executes its catalytic function and a C-terminal polo-box domain (PBD), which engages in protein - protein interactions (PPIs) with phosphoserine (pS) and phosphothreonine (pT)-containing sequences. Although Plk1 KD-directed agents are currently in clinical trials for the treatment of cancers, issues related to cytotoxicity have arisen that may result from off-target effects. Targeting protein - protein interactions (PPIs) has emerged as an important area for anticancer therapeutic development. In the case of phospho-dependent PPIs, such as the Plk1 PBD, a phosphorylated protein residue can provide high-affinity recognition and bind to target protein hot spots. Starting from the 5-mer phosphopeptide "PLHSpT" and in collaboration with the NCI laboratory of Dr. Kyung Lee and the MIT laboratory of Dr. Michael Yaffe, we initially identified inhibitory peptides that showed from 1000- to more than 10,000-fold improved PBD-binding affinity. X-ray co-crystal structures of these peptides bound to Plk1 PBD indicated unanticipated modes of binding, which take advantage of a "cryptic" binding channel that is not present in the non-liganded PBD or engaged by the parent pentamer phosphopeptide. The cryptic pocket is accessed by means of a phenylalkyl moiety attached to the N(pi) nitrogen of the His imidazole ring. Multivalency can be a powerful means to achieve highly potent and selective ligand-protein interactions. The selectivity and affinity of protein kinase (PK) inhibitors can be greatly increased by linking an element that binds within the ATP-binding cleft together with a component that binds exterior to the cleft. When the secondary component accesses ancillary regulatory domains, the resulting ligand may be described as being intramolecular "bivalent." We have undertaken work to develop bivalent ligands, designed to simultaneously engage both KD and PBD regions of Plk1. This has resulted in bivalent constructs exhibiting more than 100-fold Plk1 affinity enhancement relative monovalent PBD-binding ligands, which had until this time, exhibited among the highest PBD-binding affinities yet reported. Startlingly, and in contradiction to widely accepted notions of KD-PBD interactions, we have found that extremely high affinities can be retained even with minimal linkers between KD and PBD-binding components. In addition to significantly advancing the development of PBD-binding ligands, our findings may cause a rethinking of the structure-function of Plk1 and potential implications for the physiological roles played by this kinase. Objective Two: Tyrosyl-DNA phosphodiesterase 1 (TDP1) it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of TOP1 and the -phosphate of its DNA substrate. Blocking TDP1 function would be an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. TDP1 inhibitors would represent a new and potentially promising class of anticancer agents that could be used with TOP1 inhibitors in anticancer therapy. Although there have been reports of TDP1 inhibitors, there is a pressing need for the discovery of effective and specific TDP1 inhibitors for which there is validated binding and a defined mechanism of actions. In collaboration with the NCI laboratories of Dr. David Waugh and Dr. Yves Pommier, used an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. More recently, in collaboration with the NCI laboratory of Dr. Jay Schneekloth, we performed a TDP1 small molecule microarray screen of over 21,000 drug-like molecules in a small molecules microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified 109 hits from 21,000 compounds (0.5% hit rate) and arrived at a preferred TDP1-binding motif. Among the hits were structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines, which we demonstrated functioned as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke-Blackburn-Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. We are currently elaborating the structure of the parent SMM-derived platform by adding functionality that extends into the peptide and DNA substrate binding regions. We are using a "click"-based oxime diversification strategy that we have used successfully in several applications to optimize the binding interactions of parent ligands. A key to this approach is its ability to take a single synthetic parent construct and easily diversity it using a library of readily obtainable aldehyde reagents. In this work, we are modifying our SMM-derived platforms by adding aminooxy handles. This yielded two parent aminooxy-containing constructs. The benzoic acid moieties of these constructs are intended to bind within the catalytic site phosphoryl-binding pocket while the aminooxy groups are situated so that the resulting oxime derivatives would access the DNA or peptide substrate-binding channels. In this way, we were able to rapidly interrogate the structures of approximately 500 oxime derivatives. The most promising compounds (low micromolar IC50 values) were further derivatized to increase the chemical stability of the parent oxime linkages. Through this process, we have been able to achieve TDP1 inhibitors with nanomolar potencies. We have recently received the crystal structure of oxime-derived inhibitors bound to the TDP1 catalytic site and it appears that they bind in a fashion that is similar to what was predicted by our molecular docking studies. Going forward our goal is to derive validated inhibitors with defined binding interactions. These inhibitors will provide pharmacological tools for studying the biochemical effects of competitively inhibiting TDP1 function in cellular settings. Our work should advance the general field of TDP1 inhibitor development. A PCT patent application has recently been filed covering aspects of this technology.

定义为抗癌治疗发展的分子靶标。 PLK1在细胞分裂中起着核心作用，PLK1活性的上调似乎与几种癌症的侵略性和预后不良密切相关。该蛋白在许多癌症中过表达，其抑制作用会导致抗增生作用。 PLK1需要N末端激酶结构域（KD）的协调作用，该结构域（KD）执行其催化功能和C末端Polo-box结构域（PBD），该结构域（PBD）从事蛋白质 - 蛋白质相互作用（PPIS）与磷酸（PPIS）与磷酸酶（PS）和磷酸雌激素（PT）序列。 尽管PLK1 KD指导的药物目前正在临床试验中治疗癌症，但与细胞毒性有关的问题可能是由于靶向效应而引起的。 靶向蛋白质 - 蛋白质相互作用（PPI）已成为抗癌治疗性发育的重要领域。在磷酸依赖性PPI的情况下，例如PLK1 PBD，磷酸化蛋白残基可以提供高亲和力识别并与靶蛋白热点结合。从5-MER磷酸肽“ PLHSPT”开始，并与Kyung Lee博士的NCI实验室和Michael Yaffe博士的MIT实验室合作，我们最初确定了抑制性肽，这些抑制性肽从1000-到10,000倍至10,000倍以上改善了PBD结合亲和力。与PLK1 PBD结合的这些肽的X射线共晶结构表明意外的结合模式，它利用了非配合PBD中不存在的“隐秘”结合通道或由亲肠pentamer phosper phopphoppide粘结的。 通过连接到咪唑环的N（PI）氮的苯基烷基部分来访问隐秘的口袋。多价性可能是实现高度有效和选择性配体 - 蛋白质相互作用的有力手段。可以通过将结合在ATP结合裂缝内结合的元素与结合与裂缝外部结合的成分结合的元素来大大提高蛋白激酶（PK）抑制剂的选择性和亲和力。当次级成分访问辅助调节域时，可以将所得的配体描述为分子内“二价”。我们已经开展了二价配体的工作，旨在同时参与PLK1的KD和PBD区域。这导致了二价构建体，表现出超过100倍的PLK1亲和力增强相对单价PBD结合配体，直到这段时间才显示出最高的PBD结合亲和力之一。令人震惊的是，与广泛接受的KD-PBD相互作用概念相矛盾，我们发现即使KD和PBD结合组件之间的最小接头也可以保持极高的亲和力。除了显着推进PBD结合配体的发展外，我们的发现还可能引起PLK1结构功能的重新思考，以及对这种激酶扮演的生理角色的潜在影响。目标两个：酪酶-DNA磷酸二酯酶1（TDP1），它能够通过修复TOP1与DNA的停滞的共价复合物来降低I型拓扑异构酶（TOP1）抑制剂的抗癌作用。它通过促进TOP1的Y723残基与其DNA底物的 - 磷酸盐之间的磷酸二酯键的水解来实现这一目标。 阻断TDP1功能将是提高TOP1抑制剂和克服耐药性的有吸引力的手段。 TDP1抑制剂将代表一种新的且潜在的抗癌药物类别，可与抗癌治疗中的TOP1抑制剂一起使用。尽管有TDP1抑制剂的报道，但仍需要发现有效的特定TDP1抑制剂，这些抑制剂具有验证的结合和确定的作用机制。与David Waugh博士和Yves Pommier博士的NCI实验室合作，使用了600多个片段的X射线晶体学屏幕，以鉴定邻苯二甲酸和羟基喹啉基序的小分子变化，这些分子在TDP1催化口袋内结合。然而，这些化合物中的大多数显示出有限的（毫米）TDP1抑制作用。最近，通过与Jay Schneekloth博士的NCI实验室合作，我们在小分子微阵列（SMM）格式中进行了TDP1小分子微阵列屏幕，以使用超过21,000个类似药物的分子（SMM）格式，以便它们的能力绑定Alexa Fluor 647（AF647）（AF647） - 小标准词。屏幕从21,000种化合物（命中率为0.5％）确定了109次命中，并获得了首选的TDP1结合图案。在结构上是n，2-二苯基咪唑唑[1,2-A]吡嗪-3-胺，我们证明它们起着TDP1粘合剂和催化抑制剂的作用。然后，我们使用一锅Groebke-Blackburn-Bienayme多组分反应探索了核心杂环骨骼，并到达具有较高抑制作用的类似物。求解一个化合物子集的TDP1共晶结构在TDP1催化位点显示其结合，同时模仿底物相互作用。我们目前正在通过添加扩展到肽和DNA底物结合区域的功能来详细阐述父源性SMM衍生平台的结构。我们正在使用基于“点击”的氧电多元化策略，在几种应用中成功使用了这些策略，以优化父配体的结合相互作用。这种方法的一个关键是它可以使用可轻松获取的醛试剂库来采用单个合成父构建体并轻松多样性。在这项工作中，我们正在通过添加aminooxy手柄来修改SMM衍生的平台。这产生了两个父母含有氨基的构建体。这些构建体的苯甲酸部分旨在结合催化位点磷酸化结合口袋，而氨基氧基组则位于氨基酸基团的位置，以便所得的氧气衍生物可以访问DNA或肽底物结合通道。通过这种方式，我们能够快速审问约500氧电衍生物的结构。将最有希望的化合物（低微摩尔IC50值）进一步衍生，以提高母氧连接的化学稳定性。通过此过程，我们已经能够实现具有纳摩尔势力的TDP1抑制剂。最近，我们收到了与TDP1催化位点结合的氧电机来源抑制剂的晶体结构，看来它们以与我们的分子对接研究所预测的方式结合。未来，我们的目标是通过定义的结合相互作用得出经过验证的抑制剂。这些抑制剂将提供药理工具，用于研究竞争性抑制TDP1功能在细胞环境中的生化作用。我们的工作应该推进TDP1抑制剂开发的一般领域。最近已提交了PCT专利申请，涵盖了该技术的各个方面。